CN110367123B - Resistance identification method for sweet potato leaf curl virus disease - Google Patents
Resistance identification method for sweet potato leaf curl virus disease Download PDFInfo
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Abstract
The invention discloses a resistance identification method of sweet potato leaf curl virus, which comprises the following steps: after the variety to be identified is cultured by a virus-free test-tube plantlet and subjected to molecular detection, the stock plantlet does not contain the leaf curl virus SPLCV; performing plug rapid propagation; leaf curl virus indicates high-power propagation of plants; identifying insect-mediated inoculation of the variety detoxified tissue culture seedlings under isolation and closed conditions; identifying grafting and virus grafting of the plant under the condition of a net room; the yield comparison test comprises planting, harvesting, yield measurement and seed preservation; seeding in the second year, investigating and identifying emergence leaf curl virus manifestation rate of the variety; the cultivars were evaluated for anti- (leaf curl) virus properties. The invention relates to an identification method for resisting Sweet Potato Leaf Curl Virus (SPLCV), which utilizes the propagation law of the virus to carry out natural virus transmission, insect vector virus inoculation and grafting virus inoculation on an identified variety at different periods, and the resistance, tolerance and infection characteristics of the variety to the sweet potato leaf curl virus are determined by classifying according to the classification standard through the second-year seedbed expression of each variety, thereby providing a basic resistance source for breeding the variety resisting the sweet potato leaf curl virus.
Description
Technical Field
The invention relates to a resistance identification method of sweet potato leaf curl virus.
Background
The sweet potato is used as the seventh food crop of the world and is mainly distributed in Asia, Africa, Latin America and other regions, and the planting area of China accounts for about 80 percent of the total number of the world. But the sweet potato yield and quality are seriously influenced due to the wide existence of sweet potato virus diseases. The data show that the incidence rate of the sweet potato virus disease field variety is 100%, the sweet potato is greatly reduced in yield even in top-dead-low yield due to the virus disease in east Africa and south America, and the sweet potato virus disease is successively reported in America and Japan to cause the reduction of the yield and the quality of the sweet potato, is an important disease in sweet potato producing areas in China and is an important reason for causing the degradation and the yield reduction of the sweet potato. The infection of virus disease can reduce the yield by 30-50%, the sweet potato after virus removal can greatly improve the yield and quality, the yield of the general variety is increased by more than 20% and up to more than 200% after virus removal, and the varieties have larger difference.
The geminivirus is a single-stranded circular DNA virus with twin particle morphology in plant viruses, is one of the biggest families in the plant viruses, and can cause destructive harm to a plurality of important economic crops such as tomatoes, cotton, cassava, beans, wheat, corns, sweet potatoes and the like. The sweet potato geminivirus disease is one of important virus diseases of the sweet potatoes, the disease is in an ascending trend, and the harm is increasingly serious. The geminivirus infecting Sweet potatoes in China is mainly Sweet Potato Leaf Curl Virus (SPLCV), and the virus is accumulated in Sweet potato tissues in large quantity after being infected, so that the yield loss can reach 20% -80%, but the Sweet potato leaf curl virus is always ignored in production due to high-temperature cryptosis and is easy to diffuse along with the communication of Sweet potato germplasm resources.
The identification method of the sweet potato variety for resisting (resistant) viruses is rarely reported in China, and in the identification of the virus resistance of more than 1200 domestic and foreign germplasm resource materials stored by Xuzhou sweet potato research center reported by the Schchen-Relay (2002), the adopted method is to take a variety cell as a unit, visually observe the symptoms and severity of leaf virus diseases and divide the symptoms into five grades. Zero order: careful visual inspection of the whole plot without typical symptomatic leaves; first-stage: about 10% or less of the leaves have a few systemic symptoms; and (2) second stage: about 20% of leaves have typical symptoms; third-stage: 40-60% of leaves have typical symptoms; four stages: more than 80% of leaves have typical symptoms. And then carrying out symptomatic leaf site survey on part of the variety. The leaf position takes the 1 st fully-unfolded young leaf of the stem tip as the 1 st leaf position, the 2 nd, 3 rd, … th and nth leaf positions below the young leaf position, the toxicity carrying condition of the pinnate mottle virus of the sweet potatoes of different leaf positions of each variety is detected by a serological method, the lowest leaf position with positive is seen, the virus-free seedling and the non-virus-free control seedling of the same variety are cultivated in the same field, and the production performance is compared. Ankang (2005) grafts the identified potato seedlings to an indicator plant Ipomoea setosa, and performs the virus resistance identification of the germplasm resources by using the indicator plant showing virus-carrying symptoms. Wangshuang published a paper of identification method of resistance to SPVD for virus diseases of sweet potatoes and estimation of yield loss in plant protection (2014, volume 41, stage 2), sweet potato stems infected with SPVD are used as scions, grafting and inoculation are carried out by a side-grafting method, then transplanting are carried out in the field, grafting is carried out at the evening, relatively thick scions and stocks are selected, the scion survival rate is close to 100%, and relatively uniform and consistent morbidity is obtained after grafting. The disease index and the yield loss rate are used as resistance evaluation indexes, and the difference among varieties is obvious, which indicates that the method can be used for identifying the resistance of sweet potato varieties to SPVD. The three methods have certain feasibility for evaluating the virus resistance of partial sweet potato variety viruses, but have many problems for identifying sweet potato leaf curl viruses mainly taking insects as mediators, and two methods exist in the virus resistance of varieties: one is that the variety carries the antiviral gene itself, the other is that the insect-borne virus is reduced, and the disease-avoiding function is achieved. Therefore, the method is as close to the production reality as possible when determining the anti-leaf curl virus characteristics of the variety. The sweet potato is a vegetative propagation crop, viruses are gradually accumulated in the sweet potato body to cause diseases, and the accumulation speed is also an important index for evaluating the tolerance resistance of the variety, so that a complete identification method for the resistance of the sweet potato leaf curl virus disease is finally formed through virus-free culture, plug propagation, quantitative insect transfer, high-power rapid propagation virus seedling grafting of an indication plant and yield comparison of the variety.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a resistance identification method for sweet potato leaf curl virus.
In order to achieve the purpose, the invention adopts a resistance identification method of sweet potato leaf curl virus, which comprises the following steps:
1) and (3) quickly propagating stock seedlings of the variety to be identified without the leaf curl virus disease: taking the stem tip of a variety of potato seedlings to be tested in a tissue culture room in the last 9 months, cutting 0.1-0.3mm apical meristem under a microscope, culturing in a culture medium to obtain a plant, detecting viruses to obtain a virus-free tissue culture seedling, propagating the virus-free tissue culture seedling in a test tube to 150 plants, culturing for 35 days, and transplanting into a substrate plug tray;
2) and (3) plug rapid propagation: at the beginning of 2 months, taking 150 virus-free tissue culture test tube seedlings cultured for 35 days, dividing the virus-free tissue culture test tube seedlings into three parts, transplanting 50 seedlings into a plug tray which is 0.7cm in diameter and is added with a prepared matrix in advance, and culturing for 30 days for later use;
3) leaf roll virus indicates high-power propagation of plants: planting an indicator plant, namely the bur datura stramonium or the heart-leaf tobacco, in a sowing box, when true leaves grow out, taking a severe strain of the sweet potato leaf curl virus as a scion to carry out virus inoculation (grafting or friction), inoculating a certain amount of insect vectors (trialeurodes vaporariorum or aphid), and culturing for later use after 60 days;
4) identifying the virus-free domesticated seedling of the variety, and carrying out insect-mediated virus inoculation under the closed condition: placing the plug tray with 45 survived virus-free seedlings in a climatic chamber with the temperature of 28 ℃ and the humidity of 90 percent, simultaneously transferring 30 cultured virus-indicating plants of the Dendrolimus albomarginata or the Xinyeyan tobacco with 500 head whiteflies into the climatic chamber, and culturing for 30 days;
5) performing virus treatment under the condition of identifying plant net shed: ridging and planting in a net shed of more than 40 meshes, determining the ridge spacing and the plant spacing arrangement cells of the variety to be identified according to the actual conditions of the net shed, planting 45 virus-free domesticated seedlings in the cells, and grafting or rubbing and inoculating virus by using cultured virus-indicating plant rodgersia albuginea or heart-leaf tobacco as a material after 14 days for later use;
6) and (3) yield comparison test planting, harvesting, yield measurement and seed preservation: simultaneously planting the insect-media virus-inoculated, grafting virus-inoculated and non-virus-inoculated virus-free tissue culture seedlings of each variety to be identified into a field, setting 3 times for each 15 strains of each variety to be a cell, generally planting the strains in 6 months and 20 days, harvesting the strains in 10 months and 15 days, respectively recording the underground and overground yield of each cell, and storing the strains in the cells;
7) investigation and identification of emergence leaf curl virus manifestation rate of the variety: sampling various varieties from insect-borne virus inoculation, grafting virus inoculation and non-virus inoculation cells respectively in 3 months in the next year, randomly taking 2.5kg per cell and dividing into three parts for seeding, investigating and identifying the quantity of the disease-showing seedlings and the quantity of the disease-showing seedlings of the leaf curl virus strains of the varieties on the 30 th day of seeding, respectively recording, calculating the disease-showing rate, and evaluating the resistance of the varieties according to the average disease-showing rate;
8) evaluation of cultivar anti (resistance) to leaf roll virus properties:
resisting: the average symptom rate is below 5%;
resisting: the average symptom rate is 5 to 15 percent;
feeling: the average symptom rate is more than 15-30%;
high feeling: the average symptom rate is more than 30%.
Compared with the prior art, the invention provides the identification method for resisting the Sweet Potato Leaf Curl Virus (SPLCV), natural virus transmission, insect vector virus inoculation and grafting virus inoculation are carried out on the identified variety at different periods by utilizing the virus propagation rule, the resistance, tolerance and infection characteristics of the variety to the sweet potato leaf curl virus are determined by classifying the second-year seedbed expression of each variety according to the classification standard, and a basic resistance source is provided for breeding the variety resisting the sweet potato leaf curl virus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below. It should be understood, however, that the description herein of specific embodiments is only intended to illustrate the invention and not to limit the scope of the invention.
A resistance identification method for sweet potato leaf curl virus comprises the following steps:
1) the stock seedling is determined to be rapid propagation without the leaf curl virus SPLCV after virus-free culture and molecular detection of the variety to be identified
Carrying out stem tip detoxification culture on potato seedlings of a variety to be tested in a tissue culture room in 9 months of the last year: taking a stem tip of a variety to be tested → cutting a 0.1-0.3mm tip meristem under a microscope → culturing in a culture medium → obtaining a plant → carrying out virus detection (the detection method is according to the industrial standard NY/T402-2016) → obtaining a virus-free tissue culture seedling → propagating the virus-free tissue culture seedling in a test tube to 150 plants, culturing for 35 days and then transplanting into a substrate plug tray;
2) fast propagation of plug
Dividing 150 virus-free tissue culture test tube seedlings cultured for 35 days into three parts at the beginning of 2 months, transplanting 50 seedlings into a plug tray which is 0.7cm in diameter and is added with a prepared matrix in advance, and culturing for 30 days for later use;
3) high power propagation of leaf curl virus indicator plants
Planting an indicator plant, namely the acerola or the oriental tobacco, in a flowerpot, taking a collected severe sweet potato leaf rolling virus strain as a scion for grafting when 2 true leaves are produced, inoculating trialeurodes vaporariorum or aphid, grafting, inoculating and culturing for 60 days for later use;
4) insect-mediated inoculation under isolation and closed conditions for identifying variety virus-free tissue culture seedlings
Placing the plug tray with 45 survived virus-free seedlings in a climatic chamber (10 square meters and capable of identifying 100 varieties) at 28 ℃ and 90% of humidity, simultaneously transferring 30 cultured virus-indicating plants of rodgersia albuginea or cardiotropha to the climatic chamber (insect medium virus inoculation) for 30 days;
5) virus treatment under condition of identifying plant net room
Forming small ridges (ridge spacing is 75cm, density is 4500 plants/mu) in a 40-mesh net room, planting 45 detoxified tissue culture seedlings of the variety to be identified into the net room, and grafting or rubbing and inoculating poison by using cultured toxicity indicating plants, namely the nitraria tangutorum bobr or the soot as materials after 14 days for later use;
6) yield comparison test planting, harvesting, yield measurement and seed preservation
Simultaneously planting three virus-free tissue culture seedlings (insect medium virus inoculation, grafting virus inoculation and non-virus inoculation) of each variety to be identified into a field, wherein 15 plants of each variety are treated as a cell, the 3-time operation is repeated, the seedlings are planted in 20 days in 6 months generally, the seedlings are harvested in 15 days in 10 months, the underground and overground yield of each cell is respectively recorded, and the seedlings are stored in the cells;
7) seeding in the second year, investigating and identifying emergence rate of leaf roll virus
In 3 months in the second year, each variety is sampled from insect media virus inoculation, graft virus inoculation and non-virus inoculation districts, 2.5kg of seeds are randomly taken from each district and divided into three parts for seeding, the quantity of the leaf rolling virus strain symptomatic seedlings and the quantity of the non-symptomatic seedlings of the variety are investigated and identified on the 30 th day of seeding, and are respectively recorded, the symptom development rate is calculated, and the variety resistance is evaluated according to the average symptom development rate;
8) evaluation of the anti-leaf curl Virus Properties of the cultivars
Resisting: the average symptom rate is below 5%;
resisting: the average symptom rate is 5 to 15 percent;
feeling: the average symptom rate is more than 15-30%;
high feeling: the average symptom rate is more than 30%.
Example 1
In 2015, 2016 and 2017, carrying out anti-leaf curl virus identification on part of main sweet potato cultivars continuously, and carrying out variety identification according to the above identified resistance identification method of Sweet Potato Leaf Curl Virus (SPLCV):
culturing a virus-free test-tube plantlet of a variety to be identified, performing molecular detection on an SPLCV-free stock plantlet containing a leaf curl virus disease → rapid plug propagation → high-power propagation of a leaf curl virus indicator plant → isolation of a virus-free tissue culture plantlet of the identified variety, performing insect-mediated virus inoculation under a closed condition → performing graft virus inoculation under a plant net room identification → performing yield comparison test planting, harvesting, yield measurement, seed preservation → seed sowing in the second year, investigating and identifying the disease rate of emergence of the variety for the leaf curl virus → evaluating the anti (resistant) leaf curl virus property of the variety according to the average value of the disease rate of the leaf curl virus. The identification of each main cultivar is shown in table 1 below.
TABLE 1 identification Table of Sweet Potato Leaf Curl Virus (SPLCV) resistance
With the combination of table 1, in 14 varieties, the Shangshu No. 19, the Longshu No. 9 and the Sushu No. 8 have no leaf rolling symptom under the conditions of natural virus transmission (non-virus) and insect-mediated virus inoculation, and have about 10% of leaf rolling symptom rate under the condition of grafting virus inoculation, which indicates that the varieties are relatively resistant to diseases and is quite consistent with the production process; the Anping I, Guanjiang 87 and Xushu 32 have no or little leaf rolling symptom under the conditions of natural virus transmission and insect-mediated virus inoculation, and have about 30 percent of leaf rolling symptom rate under the condition of grafting virus inoculation, and are relatively resistant to diseases; the Yanshu No. 5, Yanshu 25, Pushu 32 and Jishu 25 belong to susceptible varieties, and the Zhenghong No. 22, Jihei No. 1 and Jishu 26 belong to highly susceptible varieties, which is very consistent with the actual situation in production.
The above description is intended to be illustrative of the preferred embodiment of the present invention and should not be taken as limiting the invention, but rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Claims (1)
1. The method for identifying the resistance of the sweet potato leaf curl virus is characterized by comprising the following steps:
1) and (3) quickly propagating stock seedlings of the variety to be identified without the leaf curl virus disease: taking the stem tip of a variety of potato seedlings to be tested in a tissue culture room in the last 9 months, cutting 0.1-0.3mm apical meristem under a microscope, culturing in a culture medium to obtain a plant, detecting viruses to obtain a virus-free tissue culture seedling, propagating the virus-free tissue culture seedling in a test tube to 150 plants, culturing for 35 days, and transplanting into a substrate plug tray;
2) and (3) plug rapid propagation: at the beginning of 2 months, taking 150 virus-free tissue culture test tube seedlings cultured for 35 days, dividing the virus-free tissue culture test tube seedlings into three parts, transplanting 50 seedlings into a plug tray which is 0.7cm in diameter and is added with a prepared matrix in advance, and culturing for 30 days for later use;
3) leaf roll virus indicates high-power propagation of plants: planting an indicator plant, namely the nitraria tangutorum hance or the heart-leaf tobacco, in a sowing box, taking a severe strain of the sweet potato leaf curl virus as a scion for inoculation when a true leaf grows out, inoculating a certain amount of entomophilus, and culturing for later use after 60 days;
4) identifying the virus-free domesticated seedling of the variety, and carrying out insect-mediated virus inoculation under the closed condition: placing the plug tray with 45 survived virus-free seedlings in a climatic chamber with the temperature of 28 ℃ and the humidity of 90 percent, simultaneously transferring 30 cultured virus-indicating plants of the Dendrolimus albomarginata or the Xinyeyan tobacco with 500 head whiteflies into the climatic chamber, and culturing for 30 days;
5) performing virus treatment under the condition of identifying plant net shed: ridging and planting in a net shed of more than 40 meshes, determining the ridge spacing and the plant spacing arrangement cells of the variety to be identified according to the actual conditions of the net shed, planting 45 virus-free domesticated seedlings in the cells, and grafting or rubbing and inoculating virus by using cultured virus-indicating plant rodgersia albuginea or heart-leaf tobacco as a material after 14 days for later use;
6) and (3) yield comparison test planting, harvesting, yield measurement and seed preservation: simultaneously planting the insect-media virus-inoculated, grafting virus-inoculated and non-virus-inoculated virus-free tissue culture seedlings of each variety to be identified into a field, wherein 15 plants of each variety are treated as a cell, the number of the cells is 3, the cells are planted within 6 months and 20 days, the cells are harvested within 10 months and 15 days, the underground and overground yield of each cell is respectively recorded, and the cells are divided for storage;
7) investigation and identification of emergence leaf curl virus manifestation rate of the variety: sampling various varieties from insect-borne virus inoculation, grafting virus inoculation and non-virus inoculation cells respectively in 3 months in the next year, randomly taking 2.5kg per cell and dividing into three parts for seeding, investigating and identifying the quantity of the disease-showing seedlings and the quantity of the disease-showing seedlings of the leaf curl virus strains of the varieties on the 30 th day of seeding, respectively recording, calculating the disease-showing rate, and evaluating the resistance of the varieties according to the average disease-showing rate;
8) evaluating the anti-and anti-leaf curl virus characteristics of the varieties:
resisting: the average symptom rate is below 5%;
resisting: the average symptom rate is 5 to 15 percent;
feeling: the average symptom rate is 15-30%;
high feeling: the average symptom rate is more than 30%.
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| CN112522359A (en) * | 2020-12-09 | 2021-03-19 | 广东省农业科学院作物研究所 | Resistance identification method for sweet potato scab for stem tip vegetables |
| CN113396712A (en) * | 2021-06-23 | 2021-09-17 | 河北省农林科学院植物保护研究所 | Single-leaf grafting inoculation method for tomato yellow leaf curl virus and tomato chlorosis virus |
| CN113767805A (en) * | 2021-08-27 | 2021-12-10 | 中国农业科学院果树研究所 | Method for identifying apple fire blight resistance by using field plot |
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